Bacillus subtilis Bacteriophage SPP1 DNA Packaging Motor Requires Terminase and Portal Proteins

Abstract

Initiation of headful packaging of SPP1 DNA concatemers involves the interaction of the terminase, G1P and G2P, and the portal protein, G6P. G1P, which specifically recognizes the non-adjacent pacL and pacR subsites and directs loading of G2P to pacC, interacts with G6P. G2P, which has endonuclease, DNA binding, and ATPase activities, interacts with G1P and does it transiently with G6P. The stoichiometry of G1P on the G1P⅐G2P complex promotes the transition from a G2P endonuclease to an ATPase. G6P does not alter the endonuclease activity of G2P. Both G1P and G6P, which do not have endogenous ATPase activity, synergistically enhance and modulate the ATPase activity of G2P. Based on these results, we propose a model in which the modulation of the ATPase and endonuclease activities of G2P accounts for the role of the terminase in headful packaging.Many cellular processes require the action of a biological motor protein that converts chemical energy into mechanical force or directional movement. Packaging of viral head-to-tail concatemeric dsDNA 1 into viruses involves the specific interaction of virus DNA with the pre-assembled procapsid and subsequent translocation of the former into the latter, by the action of a DNA translocase, to render a highly condensed structure (1-4). DNA translocases are molecular motor proteins that use the energy of nucleoside triphosphate hydrolysis to package concatemeric dsDNA onto an empty procapsid. This is a common feature shared by the DNA packaging machinery of many bacterial, pox, and herpes viruses. Two general modes for packaging of concatemeric dsDNA into the capsid of a bacterial virus (also termed bacteriophage or phage) have been proposed. The first implies a site-specific packaging in which the recognition sequence (termed cos in phage ) plays an important role in initiation and termination of DNA encapsidation. This packaging process, which generates unit-length encapsidated molecules, is well characterized in phages , T3, and T7 (1-4). The second mode implies headful packaging, in which the encapsidation initiates at a specific site in the genome (termed pac in phage SPP1), but with the capacity of the procapsid playing a predominant role in the termination step. The sequential headful packaging mechanism generates a heterogeneous population of terminally redundant and partially circularly permuted DNA molecules as in the cases of SPP1, P1, P22, or in the case of T4 whose DNA is totally permuted and terminally redundant (Refs. 1 and 3 and Fig. 1).Bacillus subtilis phage SPP1 replication results in the formation of large head-to-tail concatemeric dsDNA. To initiate DNA packaging, the terminase, composed of small G1P and large G2P subunits, recognizes and cleaves the concatemeric DNA within the pac site (see Refs. 5 and 6 and Fig. 1). The SPP1 packaging site is divided into three discrete subsites: pacL, defining the non-encapsidated or left DNA end, pacC, the cleavage site, and pacR, the encapsidated or right DNA end (Refs. 7 and 8 and Fig. 1A). The terminase, bound to...

The high‐resolution functional map of bacteriophage SPP1 portal protein

“…5A). Several parts of the protein are thus involved in the mechanism regulating how the level of DNA headfilling is sensed and how this information is communicated to the headful endonuclease that cuts the DNA, an activity that is most probably carried out by the terminase large subunit (Tavares et al, 1995;Camacho et al, 2003).…”

“…The ensemble of the results suggest that, under certain conditions, defects in the DNA packaging machinery might promote headful cleavage but that a specific headful sensor system that measures the level of DNA headfilling is operative. This sensor recognizes when a threshold amount of DNA is reached inside the viral capsid and triggers by an unknown mechanism the headful endonucleolytic cleavage that is achieved most probably by the terminase (Orlova et al, 1999;Camacho et al, 2003).…”

“…Previous studies have shown that gp6 ensures the assembly of a procapsid structure with the correct size (Dröge et al ., 2000), that it targets the minor protein gp7 to the procapsid , that it modulates the ATPase activity of the terminase large subunit gp2 (Camacho et al ., 2003) and that it plays an essential role in the sizing of the DNA packaged (Tavares et al ., 1992). Gp6 has an overall architecture similar to that found for other portal proteins.…”

The 1.58 Å resolution structure of the DNA-binding domain of bacteriophage SF6 small terminase provides new hints on DNA binding

“…The pac site is used only once per approximately every four packaging events, generating a heterogeneous population of terminally redundant and circularly permuted viral chromosomes (Tavares et al, 1992). In the first packaging event the non-encapsidated end of pacL is degraded by nucleases, while the rest of the DNA that contains pacR is encapsidated (Tavares et al, 1992;Camacho et al, 2003) until the capsid is full.…”

Crystal structure of the DNA-recognition component of the bacterial virus Sf6 genome-packaging machine

“…These DNA-recognition components are small proteins of ∼140-210 amino acid residues in tailed bacteriophages, whereas they are large proteins with ∼700-900 amino acid residues in herpesviruses, which presumably encode additional biological functions such as interactions with other viral or host factors (11). The terminase DNA-recognition components of bacteriophages T4, T7, SPP1 and P22 have been reported to form oligomers (12)(13)(14)(15)(16). In bacteriophage lambda, the two components of terminase, gpNu1 and gpA, formed heterooligomers (17).…”